Neonatal adiposity is associated with microRNAs in adipocyte-derived extracellular vesicles in maternal and cord blood

Background Maternal body size, nutrition, and hyperglycemia contribute to neonatal body size and composition. There is little information on maternal-fetal transmission of messages which influence fetal growth. We analyzed adipocyte-derived small extracellular vesicular (ADsEV) microRNAs in maternal and cord blood to explore their adipogenic potential. Methods We studied 127 mother-neonate pairs (51 lean and 76 adipose neonates, in 68 NGT and 59 GDM pregnancies). Adiposity refers to the highest tertile (T3) of sum of skinfolds in neonates of normal glucose tolerant (NGT) mothers, lean to the to lowest tertile (T1). ADsEV miRNAs from maternal and cord blood samples were profiled on Agilent 8*60K microarray. Differential expression (DE) of ADsEV miRNAs in adipose vs. lean neonates was studied before and after adjustment for maternal gestational diabetes mellitus (GDM), adiposity, and vitamin B12-folate status. Results Multiple miRNAs were common in maternal and cord blood and positively correlated. We identified 24 maternal and 5 cord blood miRNAs differentially expressed (p ≤ 0.1) in the adipose neonate group, and 19 and 26 respectively, in the adjusted analyses. Even though DE miRNAs were different in maternal and cord blood, they targeted similar adipogenic pathways (e.g., the forkhead box O (FOXO) family of transcription factors, mitogen-activated protein kinase (MAPK) pathway, transforming growth factor beta (TGF-β) pathway). Maternal GDM and adiposity were associated with many DE ADsEV miRNAs. Conclusion Our results suggest that the ADsEV miRNAs in mothers are potential regulators of fetal adiposity. The expression and functionality of miRNAs appears to be influenced by maternal adiposity, hyperglycemia, and micronutrient status during pregnancy.


Introduction
Women undergo many physiological adaptations during the gestational period to promote healthy fetal growth 1 . The developing fetus depends on transfer of nutrients from the mother for the growth of its developing tissues and organs. Exposure of the fetus to overnutrition (maternal hyperglycemia) or undernutrition (maternal macro-and micro-nutrient de ciencies) during the intra-uterine development could create a 'memory' of malnourishment which is proposed to "program" its risk for future adiposity, diabetes, and cardiovascular diseases 2 . This is the science of the Developmental Origins of Health and Disease (DOHaD) 3 .
The Pune birth cohort studies (Pune Children's Study (PCS) and Pune Maternal Nutrition Study (PMNS)) provided signi cant insight into the fetal origins of elevated risk for chronic disease 4 . The PMNS described the unique 'thin-fat' Indian baby which has lower birth weight and lean mass, but comparable fat mass, to a European baby 5 . The thin-fat Indian baby also has higher leptin and insulin but lower adiponectin concentrations in the cord blood 6 . This neonatal 'thin-fat' phenotype is thought to originate in multi-generational maternal undernutrition and is aggravated by maternal glycemia 7 . This phenotype continues into postnatal life and contributes to the higher cardiometabolic risk in Indians compared to Europeans 8 . In the Pune studies, low birth weight was associated with childhood insulin resistance, and a maternal imbalance of low vitamin B12 and high folate status was associated with high adiposity and insulin resistance in the offspring 9,10 . Prediabetes in 18-year-old offspring was predicted by poor linear growth in utero and by high normal glycemia in the mother during pregnancy.
Understanding the molecular pathways leading to fetal adiposity are of interest to the 'primordial' prevention of diabetes and other cardiometabolic diseases. Several mechanisms, such as genetics, epigenetics, nutrients, and pro-in ammatory cytokines are potential drivers of this intergenerational in uence 11 . Individually, they may explain only a small proportion of the effect. For instance, genetic markers from genome wide association studies account for less than 15% of the overall observed variance in the obesity phenotypes 12,13 . More recently small extracellular vesicles (sEV), sometimes called exosomes, came into focus as potential mediators of intergenerational transfer due to their ability to cross the placenta, the rise in sEV number in the maternal circulation during pregnancy and their important role in cell-to-cell communication 14,15 . sEVs are endocytic vesicles with a lipid bilayer, are released by a wide range of cell types and can transfer molecular cargo (e.g., proteins, nucleic acids, lipids) from the original secreting cell to a recipient cell in a paracrine or endocrine fashion 16 . More speci cally, adipocyte-derived sEVs (ADsEV) have been shown to be a major source of circulating microRNAs (miRNAs) which can regulate gene expression in distant tissues 17,18 . We have previously demonstrated that ADsEV miRNA contents are altered by obesity and normalized following signi cant weight-loss (i.e., from bariatric surgery) 19,20,21 . Further, ADsEVs from individuals with obesity alter macrophage cholesterol e ux and gene expression in vitro 22 . Given the important role of ADsEVs in obesity-related co-morbidities 23,24 , our objective was to explore ADsEV miRNAs as potential mediators of maternal-to-fetus communication and transfer of intergenerational risk for adiposity. In this analysis, we examined differences in miRNA pro les of ADsEVs isolated from maternal and cord blood plasma samples collected at delivery in pregnancies with adipose and lean neonates, with the hypothesis that ADsEV miRNAs will enhance adipogenic pathways.

Methods
Cohort description: The 'Intergenerational programming of diabesity in offspring of women with gestational diabetes mellitus' (InDiaGDM) study investigated the impact of maternal hyperglycemia on the risk of diabesity (diabetes + adiposity) in the offspring and the modifying in uence of vitamin B12 and folate status 25  Sample processing randomization and blinding:To avoid unintentional sampling bias, the sample IDs were randomly ordered ve times and then mother-neonate pairs were grouped into batches of eight pairs (N=16). Laboratory staff were provided with a blinded (i.e., no group information or metadata) processing order for samples. Statistical analyses:Sample size and power calculations were done using "ssize" package in R to measure a reliable fold change of 2.0 in miRNA expression in the adipose group with respect to lean at a signi cance level of 0.05. A total of 120 individuals (~60 in each group) would provide a power of 80%.
Demographic characteristics: Normally distributed data are presented as mean (SD) and skewed data as median (25 th and 75 th percentiles). The signi cance of differences between groups was tested by Student's t test (if normally distributed) or Mann-Whitney test (if skewed).
MicroRNA data: Raw microRNA expression data was log2 transformed and normalized using the 90th Percentile shift normalization method in GeneSpring GX (Version 14.5) software. MiRNAs that showed true signal in at least 10% of samples were selected for further analysis. To check if the average expression of all ADsEV miRNAs was upregulated or downregulated in the adipose group, mean z-scores of miRNA expression values were calculated for all miRNAs from lean and adipose groups in maternal plasma and cord blood and the distribution of z-scores between the groups were compared. We also investigated if the ADsEV miRNA expression in maternal plasma samples correlated with those from cord blood samples by applying a Spearman correlation test.
The central objective of the study was to compare differential expression of ADsEV miRNAs in maternal plasma and cord blood of adipose (T3) and lean neonates (T1). Differential expression of miRNAs between groups was calculated by Student's T test for those normally distributed and by Mann-Whitney test for skewed. Fold changes were calculated by formula: FC = average (miRNA expression in adiposemedian expression of miRNA in lean). This was done in 2 steps: 1. Without adjusting for confounding variables and 2. with adjusting for confounding variables (maternal GDM status, adiposity, and maternal vitamin B12 and folate status for maternal samples, and additionally neonatal gender for cord blood samples) bymultiple linear regression analysis. We used a discovery signi cance of p≤0.1 and a FC≥|1.2| and represented it as a volcano plot generated using ggplot2 25 package in R.
Functional analyses:Differentially expressed miRNAs with p-value≤0.1 were selected for miRNA target gene and functional enrichment using Mienturnet 27 software (http://userver.bio.uniroma1.it/apps/mienturnet/; March 2019). Target enrichment was done using miRTarBase 8.0 and the functional enrichment was conducted by using the KEGG database. This tool computationally maps the given list of miRNAs to their target genes/proteins in their reference database (KEGG/miRTarBase) and fetches a list of target genes that are computationally predicted and/or experimentally con rmed. Relevant overrepresented classes of annotated genes with at least 1 miRNAtarget interaction were selected for functional enrichment and are studied. MicroRNAs targeting biological pathways belonging to the following KEGG pathway categories are not shown because they are of no direct interest in this analysis: cancer, bacterial/viral/parasitic infectious diseases, excretory, immune and nervous system, immune and neurodegenerative diseases, environmental adaptation, aging, substance abuse, and drug resistance. Pathways with FDR p-value≤0.05 and related to adipogenesis, adipocyte differentiation and adipocyte signaling were considered relevant from the KEGG pathway categories like cell growth and development, signal transduction, metabolism, endocrine and cardiovascular health, digestive systems and studied in detail. Fold changes of each DE miRNA in adipose group are shown in a barplot. MiRNAs and their targeted genes are represented as a network plot, generated using ggnet 28 package in R. A bubble plot of the functionally enriched miRNAs was generated using ggplot2 29 , where colour and size of each bubble stands for its FDR p-value and the number of targeted genes, respectively. Adipogenesis related pathways targeted by both maternal and cord blood DE miRNAs are highlighted using a Sankey diagram generated using SankeyMatic 30 software.
Differential expression of miRNAs in relation to maternal conditions (hyperglycemia and adiposity during pregnancy):To check the in uence of maternal conditions (i.e., hyperglycemia and adiposity during pregnancy) on ADsEV miRNA pro le, differential expression analysis was carried out in both maternal and cord blood ADsEV miRNA pro les between following groups: 1. GDM and NGT (unadjusted and adjusted for confounders: maternal adiposity, B12 and folate status) and 2. maternal adiposity (below and above median) (unadjusted and adjusted for confounders: GDM, B12 and folate status). The lists of DE miRNAs from the unadjusted and adjusted analyses were combined and duplicates were removed. DE miRNAs from both analyses were subjected to functional enrichment in Mienturnet and adipogenesis related pathways were screened. A Venn diagram was made using jvenn 31 software to compare the adipogenesis related microRNAs from the three analyses: 1. Neonatal adiposity, 2. Maternal GDM, and 3. Maternal adiposity in both maternal and cord blood.

Results
The InDiaGDM study enrolled 413 women after applying inclusion and exclusion criteria. Of these, 330 women delivered at study centers. Two hundred and seventy-nine mother-neonate pairs had the necessary data and matching maternal plasma and cord blood samples. From these, guided by a power calculation, we randomly selected 127 mother-neonate pairs, including 51 lean and 76 adipose neonates, for ADsEV miRNA analyses (supplementary gure 1). There were no signi cant differences between the included (N=127) and excluded (N=152) mothers with respect to age, parity, weight, and BMI, or in their neonates with respect to gender, gestational age at the time of birth, and birth weight (supplementary  table 2).
Characteristics of mothers and neonates:Mothers were a median (IQR) age of 27 (5) years old, 155 (7) cm tall, and had a BMI of 27.1 (6.6) kg/m 2 at 28 weeks' gestation.Of the 127 mothers, 68 were NGT and 59 were diagnosed with GDM. The mothers of adipose neonates had higher weight, BMI, SSF, and fasting glucose compared to mothers of lean neonates but similar levels of total and HDL cholesterol, triglycerides, vitamin B12, and folate (Table 1). Adipose neonates had higher birth weight, length, SSF, and placental weight compared to lean neonates ( Table 1). Mothers of adipose neonates had higher prevalence of adiposity and GDM.
Differential expression of ADsEV miRNAs in maternal plasma and cord blood: Of the 2549 miRNAs pro led in the Agilent 8*60k microarray, 742 miRNAs were detected in at least 1 sample. We chose miRNAs which were expressed in at least 10% of samples for further analysis (139 miRNAs in maternal and 144 in cord blood) (supplementary gure 2). We found that 118 miRNAs were common in maternal and cord blood. Of these, 74 (63%) were positively and signi cantly correlated (p≤0.05). Comparison of mean z-score of all miRNAs in the two groups showed average miRNA expression to be lower in the adipose group in both maternal and cord blood (supplementary gure 3).
Comparison between the adipose and lean neonate groups identi ed 24 miRNAs differentially expressed in their mothers at p≤0.  Figure 1). After combining the lists of DE miRNAs in unadjusted and adjusted analyses and removing duplicates, 39 miRNAs in maternal and 29 miRNAs in cord blood were found to be differentially expressed in adipose group.
The fold changes are shown in Table 2 and Figures 2 and 3. Further screening based on KEGG ontology revealed 4 miRNAs to be targeting genes involved in pathways related to adipogenesis.
We used a Sankey diagram ( gure 4) to depict the inter-relationships between differentially expressed miRNAs in maternal and cord blood (of adipose neonates) and their targeted adipogenic pathways. The highlighted pathways included adipocytokine signaling, FoxO signaling, MAPK signaling, Hedgehog signaling, Wnt signaling, insulin signaling, AGE-RAGE signaling, cAMP signaling, JAK-STAT signaling, Ras signaling, and TGF-beta signaling.
We compared differential expression of ADsEV miRNAs in plasma of adipose vs. non-adipose and in GDM vs NGT mothers and in the cord blood of their neonates. We found 127 DE ADsEV miRNAs in adipose and 106 in GDM mothers, and 57 and 82 in the cord blood, respectively. Of these, 25 miRNAs in the adipose and 19 in the GDM mothers were related to adipogenesis, the corresponding number in the cord blood was 16 and 17 respectively. Figure 5 shows that 28 miRNAs were common in maternal blood amongst adipose mothers, GDM mothers and adipose neonates and 3 of them were related to adipogenesis: hsa-miR-451a, hsa-miR-212-3p, hsa-miR-1234-3p and 13 miRNAs were common in cord blood of which only 1 hsa-miR-204-5p was related to adipogenesis based on KEGG ontology.

Discussion
There are few reports of ADsEV miRNAs in mother-neonate pairs. We investigated the association between ADsEV miRNAs in maternal and cord blood with neonatal body composition to understand their potential in uence on neonatal adiposity. A large majority (~85%) of ADsEV miRNAs in maternal and cord blood were common, and two-thirds of these were positively correlated suggesting a crosstalk between maternal adipose and neonatal tissue. Maternal adiposity and hyperglycemia, two major determinants of neonatal adiposity were both associated with differential expression of a relatively large number of ADsEV miRNAs in maternal and fetal circulation, a fth of these were functionally related to adipogenesis. In the maternal circuit, these may represent adipose tissue dysfunction (i.e., adiposopathy) and have the potential to in uence fetal growth and body composition.
Our primary analysis was to investigate maternal and cord blood miRNAs associated with neonatal adiposity. The tables 2A and 2B and the Sankey gure ( Figure 4) show that the differentially expressed miRNAs were different in maternal and cord blood (except hsa-miR-212-3p which was common). Interestingly, they targeted the genes well-known to in uence cellular signaling pathways related to adipogenesis. (FoxO, MAPK, Insulin, AGE-RAGE signaling, etc.) ( gure 4). Literature survey revealed experimental evidence of the involvement of some of these miRNAs in aspects of adipogenesis. Maternal miRNAs hsa-miR-483-3p (FC= 3.99 in adipose group) and hsa-miR-212-3p (FC=0.282 in adipose group) have been associated with lipid accumulation and adipocyte differentiation in low-birth-weight adult humans 32 , and with increased subcutaneous adipose tissue in ammation 33 , respectively. In an earlier study we found that hsa-miR-212-3p is associated with increased risk of glucose intolerance in young adult PMNS female participants 34 . Fetal miRNAs hsa-miR-204-3p (FC=9.87 in adipose group) and hsa-miR-342-3p (FC=-0.098) have been shown to promote adipogenic differentiation of mesenchymal stem cells 35,36 and hsa-miR-15a (FC= -0.912) to regulate intramuscular adipogenesis 37 . Given that neonates have a substantial proportion of brown adipose tissue (BAT) and that Indians have lower BAT compared to Europeans, we investigated if any of these DE miRNAs have originated in the BAT. Intriguingly, hsa-miR-92a-3p (FC= -0.36) and hsa-miR-181 (FC= 0.71 in adipose group) which are associated with brown fat activity 38,39 are found to be differentially expressed only in cord blood, re ecting active brown fat activity in neonates. Together our results re ect a possible adipocyte dysfunction in mothers of adipose neonates and their role in fetal adipogenesis. Our results suggest a signaling role for maternal tissue speci c miRNAs in in uencing development of corresponding neonatal tissue, a unique example of maternal-fetal communication.
Growth and development of the fetus depends on coordinated timely expression of genes which is epigenetically regulated 40 . Body composition of the fetus is probably determined from very early pregnancy when the fate of the three germ layers is decided (gastrulation). Adipogenesis involves 'commitment' of mesenchymal stem cells to develop as pre-adipocytes which subsequently differentiate into adipocytes 41 . Several speci c genes regulate these steps and the expression of these genes in turn is regulated by DNA methylation, histone modi cations and speci c miRNAs 42,43,44 . The role of DNA methylation in adipogenesis has been studied 45,46 but there is little information on the other epigenetic mechanisms (i.e., miRNA regulation). Light microscopic studies in human abortuses demonstrate that pre-adipocyte to adipocyte differentiation happens between 19 to 23 weeks, after which period only adipocyte size increases by lipid accumulation 7 . Two major maternal determinants of these processes are maternal nutrition (both macro-and micro-nutrients) and her metabolism (diabetes and associated metabolic abnormalities) 47 . There is only scant information on how these two stimuli in uence molecular adipogenesis in the baby, though insulin secretion by fetal pancreas is an important mechanism (Abigail Fouden). Maternal hyperglycemia promotes fetal hyperinsulinemia (Pedersen's hypothesis) 48 , lipids and amino acids probably work similarly (Freinkel's fuel mediated teratogenesis) 49 . In our novel approach we have investigated the possible involvement of maternal ADsEV miRNAs in in uencing fetal adiposity and suggest that DE ADsEV miRNAs in both the maternal and neonatal circulation could in uence neonatal adiposity through regulating various cellular signaling pathways which are well-known to be involved in different aspects of adipogenesis. Though our investigation does not allow us to con rm maternal origin of ADsEVs in fetal circulation, many studies have demonstrated maternal-fetal transfer of EVs.
Our study is perhaps the rst of its kind to investigate association of maternal ADsEV miRNAs and neonatal adiposity. This is a novel approach to fetal growth and development and may provide useful leads into possible molecular mechanisms of maternal-fetal communication. An additional strength is a reasonable number of mother-baby dyads in each group in a population that is characterized by adipose newborns (thin-fat Indian babies). One of the limitations is that the maternal ADsEV miRNAs were measured only at the time of delivery and thus after neonatal adiposity is established; we will investigate maternal ADsEVs in early pregnancy to expand our ndings. It is also true that the current design does not provide conclusive proof of transfer of maternal ADsEVs and miRNAs to fetal circulation, and therefore our results are to be considered hypothesis generating. We will investigate this by HLA typing of ADsEVs to track their origin and presence of sex speci c miRNAs. It will also be interesting to pursue adiposity and metabolic outcomes in these children in relation to cord blood ADsEVs. External validity of our ndings will come only from additional studies in other populations.
In summary, we demonstrate differential expression of ADsEV miRNAs both in maternal and neonatal circulation in pregnancies with adipose neonates. These miRNAs targeted different aspects of adipose tissue function and adipogenesis, supporting a possible role in fetal adipogenesis. Studies in other populations and in early pregnancy will improve our understanding of maternal-fetal communication which in uences fetal body composition. The raw microarray data is available at Gene Expression Omnibus (Accession ID: GSE217933). The demographic, and phenotypic data is available with the authors and can be made available for a reasonable request following institutional clearances.

Funding
The work was supported by InDiaGDM grant of the Department of Biotechnology, New Delhi, India (BT/IN/Denmark/02/CSY/2014) and National Institute of Health (NIH), the U.S government (R21HD094127-01).

Acknowledgement
The authors thank Deepa Raut, Neelam Memane, Sayali Wadke and Rajashree Kamat for their help in processing cord blood samples and laboratory assays. Madhura Deshmukh for help in study coordination. Mrs. Pallavi Yajnik and Rasika Ladkat for administrative help. Dr. Leelavati Narlikar for her guidance in data analysis. Dr. Satyajit Rath for his advice in writing t discussion. We also thank the coinvestigators of the InDiaGDM study Dr. Giriraj Chandak, Kalyanaraman Kumaran, Dr. Gundu Rao for their valuable contribution in conducting the InDiaGDM study.   Figure 1 Differentially expressed miRNAs between lean Vs adipose groups in maternal and cord blood samples.    Complex interplay of DE ADsEVs miRNAs from maternal and cord blood in adipogenesis related pathways The Sankey diagram shows the DE miRNAs in maternal and cord blood of adipose neonates and their targeted pathways related to adipogenesis. The diagram highlights that though the differentially expressed miRNAs were different in maternal and cord blood, they targeted similar adipogenesis-related pathways. The numbers represent the total number of targeted genes.